Extraction and analysis of high-quality chloroplast DNA with reduced nuclear DNA for medicinal plants.

BACKGROUND: Obtaining high-quality chloroplast genome sequences requires chloroplast DNA (cpDNA) samples that meet the sequencing requirements. The quality of extracted cpDNA directly impacts the efficiency and accuracy of sequencing analysis. Currently, there are no reported methods for extracting cpDNA from Erigeron breviscapus. Therefore, we developed a suitable method for extracting cpDNA from E. breviscapus and further verified its applicability to other medicinal plants. RESULTS: We conducted a comparative analysis of chloroplast isolation and cpDNA extraction using modified high-salt low-pH method, the high-salt method, and the NaOH low-salt method, respectively. Subsequently, the number of cpDNA copies relative to the nuclear DNA (nDNA ) was quantified via qPCR. As anticipated, chloroplasts isolated from E. breviscapus using the modified high-salt low-pH method exhibited intact structures with minimal cell debris. Moreover, the concentration, purity, and quality of E. breviscapus cpDNA extracted through this method surpassed those obtained from the other two methods. Furthermore, qPCR analysis confirmed that the modified high-salt low-pH method effectively minimized nDNA contamination in the extracted cpDNA. We then applied the developed modified high-salt low-pH method to other medicinal plant species, including Mentha haplocalyx, Taraxacum mongolicum, and Portulaca oleracea. The resultant effect on chloroplast isolation and cpDNA extraction further validated the generalizability and efficacy of this method across different plant species. CONCLUSIONS: The modified high-salt low-pH method represents a reliable approach for obtaining high-quality cpDNA from E. breviscapus. Its universal applicability establishes a solid foundation for chloroplast genome sequencing and analysis of this species. Moreover, it serves as a benchmark for developing similar methods to extract chloroplast genomes from other medicinal plants.

The Current Developments in Medicinal Plant Genomics Enabled the Diversification of Secondary Metabolites' Biosynthesis.

Medicinal plants produce important substrates for their adaptation and defenses against environmental factors and, at the same time, are used for traditional medicine and industrial additives. Plants have relatively little in the way of secondary metabolites via biosynthesis. Recently, the whole-genome sequencing of medicinal plants and the identification of secondary metabolite production were revolutionized by the rapid development and cheap cost of sequencing technology. Advances in functional genomics, such as transcriptomics, proteomics, and metabolomics, pave the way for discoveries in secondary metabolites and related key genes. The multi-omics approaches can offer tremendous insight into the variety, distribution, and development of biosynthetic gene clusters (BGCs). Although many reviews have reported on the plant and medicinal plant genome, chemistry, and pharmacology, there is no review giving a comprehensive report about the medicinal plant genome and multi-omics approaches to study the biosynthesis pathway of secondary metabolites. Here, we introduce the medicinal plant genome and the application of multi-omics tools for identifying genes related to the biosynthesis pathway of secondary metabolites. Moreover, we explore comparative genomics and polyploidy for gene family analysis in medicinal plants. This study promotes medicinal plant genomics, which contributes to the biosynthesis and screening of plant substrates and plant-based drugs and prompts the research efficiency of traditional medicine.

The presence and transfer characteristics of aflatoxins in medicinal herbs: From raw materials to edible dispensing granules.

In this study, a sensitive and accurate immunoaffinity columns coupled with high-performance liquid chromatography method was established to monitor the presence of aflatoxins-aflatoxin B1 , aflatoxin B2 , aflatoxin G1 , and aflatoxin G2 -in different medicinal herbs. The proposed method was found to be suitable for the detection of aflatoxins in eight kinds of herbs and their corresponding granules. Two batches of Arecae semen were positive for aflatoxins, with high residue levels of different aflatoxins. To better understand the presence and transfer of aflatoxins during the formulation of dispensing granules from the herbs, the aflatoxins-free herbs were artificially inoculated with Aspergillus flavus to explore aflatoxins production. Both aflatoxin B1 and aflatoxin B2 were detected in all inoculated samples, while aflatoxin G2 was only detected in Astragali radix samples. Additionally, the presence of aflatoxin B1 was extremely high compared to other aflatoxins. More specifically, the transfer rate of the aflatoxin B1 and the total aflatoxins from original herbs to granules were both approximately 40%. These findings indicated that the preparation of herbs into dispensing granules reduced the content of aflatoxins. The high-level presence of aflatoxins in inoculated herbs indicated that attention is needed to the safety of A. flavus-contaminated herbs.

[Resistance of different ecotypes of Gastrodia elata to tuber rot].

Tuber rot has become a serious problem in the large-scale cultivation of Gastrodia elata. In this study, we compared the resistance of different ecotypes of G. elata to tuber rot by field experiments on the basis of the investigation of G. elata diseases. The histological observation and transcriptome analysis were conducted to reveal the resistance differences and the underlying mechanisms among different ecotypes. In the field, G. elata f. glauca had the highest incidence of tuber rot, followed by G. elata f. viridis, and G. elata f. elata and G. elata f. glauca×G. elata f. elata showed the lowest incidence. Tuber rot showcased obvious plant source specificity and mainly occurred in the buds and bottom of G. elata plants. After infection, the pathogen spread hyphae in host cortex cells, which can change the endophytic fungal community structure in the cortex and parenchyma of G. elata. G. elata f. glauca had thinner lytic layer and more sugar lumps in the parenchyma than G. elata f. elata. The transcription of genes involved in immune defense, enzyme synthesis, polysaccharide synthesis, carbohydrate transport and metabolism, hydroxylase activity, and aromatic compound synthesis had significant differences between G. elata f. glauca and G. elata f. elata. These findings suggested that the differences in resis-tance to tuber rot among different ecotypes of G. elata may be related to the varied gene expression patterns and secondary metabolites. This study provides basic data for the prevention and control of tuber rot and the improvement of planting technology for G. elata.

[Correlation analysis between continuous cropping obstacle of Gastrodia elata and Ilyonectria fungi and relieving strategy].

The continuous cropping obstacle of Gastrodia elata is outstanding, but its mechanism is still unclear. In this study, microbial changes in soils after G. elata planting were investigated to explore the mechanism correlated with continuous cropping obstacle. The changes of species and abundance of fungi and bacteria in soils planted with G. elata after 1, 2, and 3 years were compared. The pathogenic fungi that might cause continuous cropping diseases of G. elata were isolated. Finally, the prevention and control measures of soil-borne fungal diseases of G. elata were investigated with the rotation planting pattern of "G. elata-Phallus impudicus". The results showed that G. elata planting resulted in the decrease in bacterial and fungal community stability and the increase in harmful fungus species and abundance in soils. This change was most obvious in the second year after G. elata planting, and the soil microbial community structure could not return to the normal level even if it was left idle for another two years. After G. elata planting in soils, the most significant change was observed in Ilyonectria cyclaminicola. The richness of the Ilyonectria fungus in soils was significantly positively correlated with the incidence of G. elata diseases. When I. cyclaminicola was inoculated in the sterile soil, the rot rate of G. elata was also significantly increased. After planting one crop of G. elata and one to three crops of P. impudicus, the fungus community structure in soils gradually recovered, and the abundance of I. cyclaminicola decreased year by year. Furthermore, the disease rate of G. elata decreased. The results showed that the cultivation of G. elata made the Ilyonectria fungi the dominant flora in soils, and I. cyclaminicola served as the main pathogen of continuous cropping diseases of G. elata, which could be reduced by rotation planting with P. impudicus.

[Utilization of used fungus-growing materials of Gastrodia elata].

This study aims to explore the resource utilization of used fungus-growing materials produced in the cultivation of Gastrodia elata. To be specific, based on the production practice, this study investigated the recycling mechanism of used fungus-growing materials of G. elata by Phallus inpudicus. To screen edible fungi with wide adaptability, this study examined the allelopathic effects of Armillaria mellea secretions on P. impudicus and 6 kinds of large edible fungi and the activities of enzymes related to degradation of the used fungus-growing materials of G. elata. The results showed that P. impudicus can effectively degrade cellulose, hemicellulose, and lignin in used fungus-growing materials of G. elata. The cellulase activity of A. mellea was significantly higher than that of P. impudicus, and the activities of lignin peroxidase, polyphenol oxidase, and xylanase of P. impudicus were significantly higher than those of A. mellea, which was the important reason why A. mellea and P. impudicus used different parts and components of the used fungus-growing materials to absorb carbon sources and develop ecological niche differences. The growth of P. impudicus was significantly inhibited on the used fungus-growing materials of G. elata. The secretions of A. mellea had allelopathic effects on P. impudicus and other edible fungi, and the allelopathic effects were related to the concentration of allelopathy substances. The screening result showed that the growth and development of L. edodes and A. auricular were not significantly affected by 30% of A. mellea liquid, indicating that they had high resistance to the allelopathy of A. mellea. The results showed that the activities of extracellular lignin peroxidase, polyphenol oxidase, and xylanase of the two edible fungi were similar to those of P. impudicus, and the cellulase activity was higher than that of P. impudicus. This experiment can be further verified by small-scale production tests.

Mrgprb2 gene plays a role in the anaphylactoid reactions induced by Houttuynia cordata injection.

ETHNOPHARMACOLOGICAL RELEVANCE: Houttuynia cordata Thunb., a plant belonging to the family of Saururaceae, has been used as a traditional Chinese medicine for more than 1500 years. Because of its various pharmacological activities, it was widely used as antipyretic, detoxification, anti-inflammatory drugs. Houttuynia cordata (HC) injection was prepared using contemporary methods to extract effective components from H. cordata Thunb. However, the adverse event reports of HC injection are accumulating remarkably with the HC injection clinical applications increased. Previous studies demonstrated that the major side effects of HC injection were anaphylactoid reactions. Our work might shed the light on the role of Mas-related G-protein coupled receptor-X2 (MRGPRX2) in modulating drug-induced anaphylactoid reactions. AIM OF THE STUDY: We aimed to investigate the role of the mouse Mas-related G-protein coupled receptor B2 (Mrgprb2) (the orthologous gene of human MRGPRX2) in anaphylactoid reactions induced by HC injection. MATERIALS AND METHODS: Mrgprb2 related anaphylactoid reactions induced by HC injection were investigated by histamine/ß-hexosaminidase releasing, mast cell degranulation, and hind paw swelling assays by using a Mrgprb2 knockout mouse model. Furthermore, the transcriptomic profiles of the anaphylactoid reaction induced by HC injection was analyzed by RNA sequencing. RESULTS: Mice without Mrgprb2 exhibited significantly decreasing in mast cell degranulation, serum histamine release, and hind paw swelling degrees. The RNA sequencing results indicated that Mrgprb2 could play a pivotal role in HC injection induced anaphylactoid reaction mediated by mTOR/AMPK pathway. Intriguingly, our results showed that Mrgprb2 might involve in Compound 48/80 induced anaphylactoid reactions mediated by Reelin/E-cadherin axis, which suggested different roles of Mrgprb2 in anaphylactoid reactions induced by HC injection and C48/80. CONCLUSION: Our studies reported effects and underlying mechanisms of Mrgprb2 in the anaphylactoid reaction induced by HC injection.

[Identification of Cordyceps cicadae and Tolypocladium dujiaolongae based on ITS sequences and chemical pattern recognition method].

Based on ITS sequences, the molecular identification of Cordyceps cicadae and Tolypocladium dujiaolongae was carried out, and high-performance liquid chromatography(HPLC) fingerprint combined with chemical pattern recognition method was established to differentiate C. cicadae from its adulterant T. dujiaolongae. The genomic DNA from 10 batches of C. cicadae and five batches of T. dujiaolongae was extracted, and ITS sequences were amplified by PCR and sequenced. The stable differential sites of these two species were compared and the phylogenetic tree was constructed via MEGA 7.0. HPLC was used to establish the fingerprints of C. cicadae and T. dujiaolongae, and similarity evaluation, cluster analysis(CA), principal component analysis(PCA), and partial least squares discriminant analysis(PLS-DA) were applied to investigate the chemical pattern recognition. The result showed that the sources of these two species were different, and there were 115 stable differential sites in ITS sequences of C. cicadae and T. dujiao-longae. The phylogenetic tree could distinguish them effectively. HPLC fingerprints of 18 batches of C. cicadae and 5 batches of T. dujiaolongae were established. The results of CA, PCA, and PLS-DA were consistent, which could distinguish them well, indicating that there were great differences in chemical components between C. cicadae and T. dujiaolongae. The results of PLS-DA showed that six components such as uridine, guanosine, adenosine, and N~6-(2-hydroxyethyl) adenosine were the main differential markers of the two species. ITS sequences and HPLC fingerprint combined with the chemical pattern recognition method can serve as the identification and differentiation methods for C. cicadae and T. dujiaolongae.

Prevalence and beverage-related risk factors of gastroesophageal reflux disease: An original study in Chinese college freshmen, a systemic review and meta-analysis.

BACKGROUND: Beverage-drinking behavior could be a potential risk factor for gastroesophageal reflux disease (GERD) in young populations. However, GERD prevalence in this population has not been investigated, and beverage consumption's association with GERD remains inconclusive. This study aimed to evaluate the prevalence and beverage-related risk factors of GERD among Chinese college freshmen and in youth around the world. METHODS: A cross-sectional survey was conducted in Chinese college freshmen in September 2019 using random cluster sampling method. Participants completed questionnaires on demographic information, food intake frequency, and GER symptoms. Multivariate logistic regression models were applied to assess the association between beverages and GERD. Studies were retrieved from multiple databases for systemic review. The prevalence of GERD in young populations and beverage-related risk factors were pooled using random-effect models. KEY RESULTS: Based on the 3345 individuals who completed the questionnaires, GERD prevalence in Chinese college freshmen is 5.1%. Multivariate analysis showed students who drink green tea daily, and those who drink coffee regularly were more likely to develop GERD compared with those who never drink tea or coffee. The pooled prevalence of GERD in young populations is 18.0%, and frequent alcohol consumption is positively associated with GERD in general population. CONCLUSIONS AND INFERENCES: The prevalence of GERD in Chinese college freshmen is significantly lower than that in worldwide youth populations. Alcohol, green tea, and coffee consumption could be potential risk factors for GERD. Future large-scale epidemiological studies are warranted for reliable identification of beverage-related risk factors for GERD in young populations.

Comparative analysis of chemical constituents by HPLC-ESI-MSn and antioxidant activities of Dendrobium huoshanense and Dendrobium officinale.

Dendrobium huoshanense is a Chinese medicinal herb that has high quality and excellent efficacy. However, the chemical basis of its activity is still unclear. Of note, Dendrobium officinale is the most widely utilized among the Dendrobium species. Therefore, the current study systematically investigated the chemical constituents of methanolic extracts and different polar fractions of aqueous extracts from the two herbs by HPLC-ESI-MSn , and then compared in vitro antioxidant activities of their five different polar extracts. Consequently, 61 and 49 compounds were identified from D. huoshanense and D. officinale, respectively, of which 43 compounds were common to both species. In addition, 17 out of 22 different compounds were identified only in D. huoshanense. Moreover, the peak areas of some shared identical compounds of D. huoshanense were significantly larger than that of D. officinale. In vitro antioxidant evaluation results showed that the n-BuOH-soluble fraction of the two herbs exhibited remarkable antioxidant activities. Furthermore, the antioxidant activities of different fractions of D. huoshanense were separately superior to that of D. officinale, which may be attributed to its variable and high contents of flavonoids, bibenzyls and phenanthrenes. These results provide the evidence for the high quality and efficacy of D. huoshanense.

Prevalence and risk factors of belching disorders: A cross-sectional study among freshman college students.

OBJECTIVES: Belching disorders seriously affect quality of life; however, their prevalences and risk factors remain unknown. The aim of our study was to determine the prevalence and risk factors, particularly lifestyle factors, of belching disorders among freshman college students in central China. METHODS: A cross-sectional study was conducted in September 2019 in Huazhong University of Science and Technology (Wuhan, Hubei Province, China). The subjects were asked to complete a self-administered questionnaire for data collection, including sociodemographic information, lifestyle factors, and gastrointestinal symptoms. Belching disorder was diagnosed based on the Rome IV criteria. Univariate and multivariate logistic regression analyses were performed to determine the risk factors for belching disorders. RESULTS: A total of 3335 subjects were enrolled, and 78.26% were men. Among them, 1.95% (65/3335) reported belching disorders. Significant differences in the Pittsburgh Sleep Quality Index (PSQI), Student-Life Stress Inventory (SLSI) scores, and consumption of whole grains, black tea, coffee were found between the belching and non-belching groups. Multivariate logistic regression analysis showed that coffee consumption at least once weekly and a high total SLSI score (over mean + standard deviation) were independent risk factors for belching disorders, while intake of whole grains at least once weekly was a protective factor. CONCLUSIONS: Excessive belching is a common disorder among freshman college students in central China. Lifestyle factors, including consumption of whole grains and coffee, and stress, were associated with belching disorders. Therefore, dietary intervention may be a potential management for belching disorders.

Complete Chloroplast Genome Sequence of Erigeron breviscapus and Characterization of Chloroplast Regulatory Elements.

Erigeron breviscapus is a famous medicinal plant. However, the limited chloroplast genome information of E. breviscapus, especially for the chloroplast DNA sequence resources, has hindered the study of E. breviscapus chloroplast genome transformation. Here, the complete chloroplast (cp) genome of E. breviscapus was reported. This genome was 152,164bp in length, included 37.2% GC content and was structurally arranged into two 24,699bp inverted repeats (IRs) and two single-copy areas. The sizes of the large single-copy region and the small single-copy region were 84,657 and 18,109bp, respectively. The E. breviscapus cp genome consisted of 127 coding genes, including 83 protein coding genes, 36 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. For those genes, 95 genes were single copy genes and 16 genes were duplicated in two inverted regions with seven tRNAs, four rRNAs, and five protein coding genes. Then, genomic DNA of E. breviscapus was used as a template, and the endogenous 5' and 3' flanking sequences of the trnI gene and trnA gene were selected as homologous recombinant fragments in vector construction and cloned through PCR. The endogenous 5' flanking sequences of the psbA gene and rrn16S gene, the endogenous 3' flanking sequences of the psbA gene, rbcL gene, and rps16 gene and one sequence element from the psbN-psbH chloroplast operon were cloned, and certain chloroplast regulatory elements were identified. Two homologous recombination fragments and all of these elements were constructed into the cloning vector pBluescript SK (+) to yield a series of chloroplast expression vectors, which harbored the reporter gene EGFP and the selectable marker aadA gene. After identification, the chloroplast expression vectors were transformed into Escherichia coli and the function of predicted regulatory elements was confirmed by a spectinomycin resistance test and fluorescence intensity measurement. The results indicated that aadA gene and EGFP gene were efficiently expressed under the regulation of predicted regulatory elements and the chloroplast expression vector had been successfully constructed, thereby providing a solid foundation for establishing subsequent E. breviscapus chloroplast transformation system and genetic improvement of E. breviscapus.

[Herbal textual research on Rubi Fructus].

Rubi Fructus is a commonly used traditional Chinese medicine. The origin of Rubi Fructus is the dried fruit of Rubus chingii, a plant of the family Rosaceae, according to the 2015 edition of Chinese pharmacopoeia. There are some differences in the plant origin of Rubi Fructus in ancient herbal literature, to trace back its sources, we conducted a textual research on its origin, producing areas, quality evaluation, processing and concocting, properties, tastes and efficacy etc. based on the records of ancient herbal literatures and combined with plant morphology and related investigation. RESULTS:: showed that the variety of Rubi Fructus was more complex among ancient herbal literature, including R. coreanus, R. hirsutus, R. corchorifolius, R. foliolosus and other mixed varieties. Most scholars believe that the R. chingii has not been recorded in ancient herbal literature, while R. chingii was recorded as early as the Ming Dynasty in Compendium of materia medica through our textual research. Ancient Chinese herbs recorded that Rubi Fructus was mostly produced in Hubei, Shandong, Shanxi and Jiangsu provinces, while R. chingii mainly produced in Anhui, Jiangsu, Zhejiang, Jiangxi, Fujian and other provinces nowadays. Also, it was recorded that Rubi Fructus harvested in wheat field during May were the best. Besides, R. chingii with big, full, grain integrate, firm, yellow and green color, sour taste and impurity free possess the best quality in the contemporary. The ancient records of processing and concocting, properties, tastes and efficacy were basically the same as modern ones.These results provide the basis for the correct utilization and further development of Rubi Fructus.

[Systematic analysis on chemical constituents of Mori Cortex, mulberry root bark and its phellem layer based on HPLC-ESI-MS].

In this study, HPLC-ESI-MS and HPLC methods were established to explore the differences in the main chemical components and content of Mori Cortex with(mulberry root bark) and without(Mori Cortex) the phellem layer from both qualitative and quantitative aspects. The HPLC-ESI-MS method was used for quality analysis in positive and negative ion modes, and 33 compounds were identified in mulberry root bark, 22 compounds in Mori Cortex, and 26 compounds in phellem layer; mulberry root bark and Mori Cortex shared 22 components, and mulberry root bark has 11 unique compounds; Mori Cortex and its phellem layer shared 15 components, while Mori Cortex has 7 unique compounds. HPLC method was used to simultaneously determine 7 major constituents, including mulberroside A, chlorogenic acid, dihydromorin, oxyresveratrol, moracin O, kuwanon G, and kuwanon H, and the developed method showed good linearity(r>0.998 9) within the concentration range and the recoveries varied from 99.88% to 103.0%, and the RSD was 1.7%-2.9%. The HPLC results showed that the contents of the 7 compounds have great differences in 13 batches samples, compared with mulberry root bark, the contents of mulberroside A, chlorogenic acid, dihydromorin and moracin O of Mori Cortex were increased, while the contents of oxyresveratrol, kuwanon G and kuwanon H were decreased after peeling process. These results can provide a basis for the rationality and quality control of Mori Cortex required to remove the phellem layer.

[Comparison of active ingredients and protective effects of Dendrobium huoshanense of different growth years on acute liver injury].

The differences of the active ingredients in Dendrobium huoshanense of different growth years and their protective effects on acute liver injury were studied to provide evidence for optimizing harvest time. The contents of polysaccharides, total flavonoids and total alkaloids in D. huoshanense of different growth years were determined by UV spectrophotometry, and the contents of gigantol in D. huoshanense were determined by HPLC. C57 BL/6 mice were randomly divided into blank control group(saline), modeling group(saline), high-dose(7.5 g·kg~(-1)) and low-dose(1.25 g·kg~(-1)) groups of D. huoshanense of different growth years. Each group was intragastrically administered every day for 2 weeks. 500 mg·kg~(-1) paracetamol was injected intraperitoneally 2 h after last treatment except the control group. After 12 hours, the serum and liver tissues were collected to detect the activities of ALT and AST, and the levels of SOD and MDA. The hepatic histopathological examination was performed. The results showed that the chemical constituents of D. huo-shanense of different growth years were significantly different(P<0.05). The contents of polysaccharide and gigantol of D. huoshanense of 2 growth years were the highest. The contents of flavonoids and alkaloids of D. huoshanense of 3 growth years were the hig-hest, followed by the D. huoshanense of 2 growth years, and the lowest were that of 1 growth year. Compared with the modeling group, D. huoshanense of different growth years could decrease the activities of ALT and AST in serum. Meanwhile, the levels of MDA reduced significantly, while those of SOD increased markedly. Histopathological results suggested that all D. huoshanense samples were effective in the reduction of the necrosis of hepatocytes in different degrees. The results of the multi-component SPSS paired tests showed that polysaccharide and gigantol probably played a leading role in the liver protection effects, while D. huoshanense of 2 growth years showed the best efficacy. The optimal harvesting time of D. huoshanense is 2 growth years.

A Chromosome-Level Genome Assembly of Dendrobium Huoshanense Using Long Reads and Hi-C Data.

Dendrobium huoshanense is used to treat various diseases in traditional Chinese medicine. Recent studies have identified active components. However, the lack of genomic data limits research on the biosynthesis and application of these therapeutic ingredients. To address this issue, we generated the first chromosome-level genome assembly and annotation of D. huoshanense. We integrated PacBio sequencing data, Illumina paired-end sequencing data, and Hi-C sequencing data to assemble a 1.285 Gb genome, with contig and scaffold N50 lengths of 598 kb and 71.79 Mb, respectively. We annotated 21,070 protein-coding genes and 0.96 Gb transposable elements, constituting 74.92% of the whole assembly. In addition, we identified 252 genes responsible for polysaccharide biosynthesis by Kyoto Encyclopedia of Genes and Genomes functional annotation. Our data provide a basis for further functional studies, particularly those focused on genes related to glycan biosynthesis and metabolism, and have implications for both conservation and medicine.

Occurrence and analysis of mycotoxins in domestic Chinese herbal medicines.

For time immemorial, Chinese herbal medicines (CHMs) have been widely used in China for disease treatment and promotion of general well-being. However, in recent years, many studies have shown that mycotoxins produced by fungi could contaminate CHMs due to unfavourable pre- or post-harvest conditions, raising major concern for consumer safety. At present, there is a significant focus on developing novel mycotoxin detection methods for analysing CHMs, and numerous studies have aimed to determine which kinds of raw herbal materials are most susceptible to mycotoxin contamination. In this review, we focus on recent advances in understanding and detection of mycotoxins in domestic raw herbal materials and related products from 2000 to 2018. Aspects of mycotoxin contamination of CHMs covered in this review include common mycotoxin contaminants in CHMs, maximum mycotoxin residue limits, analytical methods for mycotoxin detection and their applications and limitations, as well as a brief discussion of the trends in ongoing research.

Lilium davidii extract alleviates p­chlorophenylalanine­induced insomnia in rats through modification of the hypothalamic-related neurotransmitters, melatonin and homeostasis of the hypothalamic-pituitary-adrenal axis.

CONTEXT: Lilium davidii var. unicolour Cotton (Lilium genus, Liliaceae) is an edible plant and a herb used in China to alleviate insomnia. OBJECTIVE: To investigate the alleviating insomnia mechanism of L. davidii (LD). MATERIALS AND METHODS: Wistar rats were intraperitoneally injected with p-chlorophenylalanine (PCPA) to establish an insomnia model. Rats were divided into six groups (n = 8): Control, PCPA, Estazolam (0.5 mg/kg), LD extract in low, medium and high doses (185.22, 370.44, 740.88 mg/kg). Serum hormone levels of the HPA axis, levels of 5-HT, NE and MT, and the expression of GABAA and 5-HT1A receptors in hypothalamus were determined. Moreover, behavioural and pathological changes in the hypothalamus were evaluated. RESULTS: After LD administration, body weight and brain coefficient increased by 2.74% and 8.22%, respectively, and the adrenal coefficient decreased by 25%, compared with PCPA group. Elevation of the serum hypothalamic-pituitary-adrenal (HPA) axis hormone CRH (11.24 ± 3.16 ng/mL), ACTH (565.87 ± 103.44 pg/mL) and CORT (44.28 ± 8.73 ng/mL) in the PCPA group was reversed after LD treatment. Furthermore, abnormal excitatory behaviour [5 min movement distance (2096.34 ± 259.51 cm), central exercise time (5.28 ± 1.08 s)] of insomnia rats in the PCPA group was also relieved. LD extract increased 5-HT and MT levels, reduced NE level in the hypothalamus, and upregulated the expression of GABAA R and 5-HT1A. Moreover, LD extract may improve the pathology of neurons in the hypothalamus. CONCLUSIONS: LD can be considered to develop health-care food or novel drugs to cope with the increasing number of insomniacs.

[Preparation of highly sensitive monoclonal antibody against aflatoxin B_1 and its application in rapid detection of contamination in Ziziphi Spinosae Semen].

A highly sensitive monoclonal antibody against aflatoxin B_1(AFB_1) was prepared and an indirect competition enzyme-linked immunosorbent assay(ic-ELISA) was established based on the antibody which was used for high-throughput and rapid screening of AFB_1 contamination in Chinese herbal medicines to ensure the safety of medication. In this study, the structure of AFB_1 was modified by improved oxime method, and the carrier protein was coupled by EDC-NHS method to obtain the complete antigen of AFB_1, which was more convenient and environmental friendly. The Balb/c female mice were immunized using increasing the immunization dose and various ways of injection, and finally the AFB_1 monoclonal antibody was prepared. The AFB_1 monoclonal antibody belongs to IgG_(2 b) immunoglobulin by identifying its immunological characteristics, and its sensitivity(IC_(50)) can reach 0.15 µg·L~(-1), and the affi-nity is 2.81×10~8 L·mol~(-1). The cross-reaction rates of AFB_2, AFG_1, and AFG_2 were 35.07%, 8.75%, and 1.15%, respectively, and there was almost no cross-reactivity with other mycotoxins. Based on the high sensitivity and specificity of the antibody, an ic-ELISA method was established and applied to the determination of AFB_1 contamination in Ziziphi Spinosae Semen. According to the matrix matching standard curve, the linear concentration range for AFB_1 was 0.05-0.58 µg·L~(-1)(R~2=0.992), the recoveries were 88.00%-119.0%, and the detection limit was 1.69 µg·kg~(-1). The AFB_1 in 33 batches of Ziziphi Spinosae Semen samples was determined by ic-ELISA, and the contamination level was 3.62-206.58 µg·kg~(-1). The linear correlation coefficient between the detection results of ic-ELISA and UHPLC-MS/MS was 0.996, and there were no false positive and false negative cases. It indicates that the established ic-ELISA is accurate and reliable, and could provide a simple and effective technique for fast screening of AFB_1 contamination in Ziziphi Spinosae Semen, and also could be considered as the reference for the detection and monitoring of AFB_1 contamination in other Chinese herbal medicines.

Frankincense and myrrh and their bioactive compounds ameliorate the multiple myeloma through regulation of metabolome profiling and JAK/STAT signaling pathway based on U266 cells.

BACKGROUND: Frankincense and myrrh are used as traditional anti-inflammatory and analgesic medicines in China. It has been reported that frankincense and myrrh have significant anti-tumor activities. The present study was designed to investigate the inhibitory efficacy of frankincense ethanol extracts (RXC), myrrh ethanol extracts (MYC), frankincense -myrrh ethanol extracts (YDC), frankincense -myrrh water extracts (YDS) and their main compounds on U266 human multiple myeloma cell line. METHODS: The inhibition effects of cell proliferation was evaluated by MTT assays. Cell culture supernatant was collected for estimation of cytokines. Western blot analysis was designed to investigate the regulatory of JAK/STAT signal pathway. In addition, cell metabolomics based on the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) had been established to investigate the holistic efficacy of frankincense and myrrh on U266 cells. Acquired data were processed by partial least-squares discriminant analysis (PLS-DA) and orthogonal projection to latent structures squares-discriminant analysis (OPLS-DA) to identify potential biomarkers. RESULTS: RXC, MYC significantly inhibited the proliferation of U266 cells at dose of 25-400 µg/mL, YDC and YDS at the dose of 12.5-400 µg/mL. 3-O-acetyl-α-boswellic acid, 3-acetyl-11 keto-boswellic acid and 11-keto-boswellic acid had the most significant anti- multiple myeloma activities in the 10 compounds investigated, therefore these 3 compounds were selected as representatives for Elisa assay and western blotting experiments. All the extracts and active compounds ameliorated the secretion of cytokines and down-regulated the expression of JAK/STAT signaling pathway-related proteins. Comparing RXC, MYC, YDC and YDS-treated U266 cells with vehicle control (DMSO), 13, 8, 7, 7 distinct metabolites and 2, 2, 3, 0 metabolic target pathways involved in amino acid metabolism, lipid metabolism, vitamin metabolism, arachidonic acid were identified, respectively. CONCLUSIONS: Taken together our results suggest that the frankincense and myrrh and their bioactive compounds inhibit proliferation of U266 multiple myeloma cells by regulating JAK/STAT signaling pathway and cellular metabolic profile.